Recent experiments provide strong evidence that the protein ductin plays an important role in craniofacial development. Ductin, also known as the 16kD subunit c of V-ATPase, functions in transmembrane transport of protons out of the cytoplasm and into vesicles or the extracellular space; ductin can probably also form gap junctions either alone or with connexins. Thus, ductin has important roles in ion flux across intra-, inter-, and extra-cellular boundaries. We recently discovered that misexpressing wildtype ductin in Xenopus embryos leads to craniofacial abnormalities, as does expressing a dominant negative ductin or injecting antiductin antibodies. The phenotypes of treated embryos, including malformed eyes, ears, and skull, look remarkably like those caused by injection of anti-connexin antibodies and misexpression of the Xenopus frizzled-3 protein. We already know that ductin functions upstream of the Sonic hedgehog signaling pathway; our new results raise the fascinating prospect that ductin and ion flux are also upstream of the Wnt-frizzled cascade during morphogenesis of the vertebrate head. We propose to explore the hypothesis that ductin-based ion flux is an early step in craniofacial morphogenesis that regulates signaling cascades via both V-ATPase dependent proton pumping and cell-cell gap junctional communication. Imaging using in situ hybridization, immuncytochemistry, and in vivo monitoring of pH and fluorescently labeled proteins will be used to characterize ductin's spatial and temporal expression pattern and to monitor the effects of ductin inhibition and misexpression. Loss and gain of function experiments using wildtype and dominant negative ductin constructs will allow us to study the relationship of ductin to downstream genetic pathways. This work will yield important and novel information about the under-studied phenomenon of biophysical control of developmental events, e.g., ion control of secreted signals, thus providing unique and valuable insight into normal morphogenesis and suggesting novel approaches to treating and preventing birth defects.